PROJECT SUMMARY Abuse of methamphetamine (METH) is a growing medical and societal problem in the US, as studies have suggested that it produces long-term neurodegenerative damage to dopamineric and serotonergic nerve terminals in multiple brain areas. This neurotoxicity has been shown to be associated with METH-mediated production of reactive oxygen species (ROS); it is believed that these initiating events result in oxidative stress with subsequent psychotic and violent behaviors in individuals. Oxygen (O2) is both the source of ROS and the terminal electron acceptor for many enzymes that are important in brain function, including those involved in the biosynthesis of the neurotransmitters dopamine, serotonin and norepinephrine that have been linked with METH abuse. Thus, alteration in cerebral tissue partial pressure of O2 (pO2) by METH abuse may directly impact homeostasis and lead to METH-induced neurotoxicity. Recently we have obtained intriguing preliminary results showing a localized dramatic decrease in cerebral tissue pO2 by EPR oximetry after acute METH administration, demonstrating the urgent need to fully define the possible heterogeneous distribution of tissue pO2 alterations in brain following METH abuse. Such tissue pO2 changes occur following stroke and traumatic brain injury and have been suggested from in vitro studies of METH-induced neurotoxicity, but cerebral tissue pO2 measurements associated with METH in vivo has not been explored, due in part, to a lack of adequate analytic methods. We propose to synthesize electron paramagnetic resonance (EPR) O2-sensitive isotopic-substituted nitroxides, e.g., spin probes that can be delivered to and distributed throughout the brain, which can map and quantify cerebral tissue O2 by EPR imaging (EPRI) after mice have received METH. In Aim 1, we will synthesize novel isotopic-substituted nitroxide-based oximetry probes for EPRI in a mouse brain and determine the most effective nitroxide in addition to the optimal conditions for the successful use of these probes as EPRI agents. In aim 2, we will obtain the temporal and spatial profile of cerebral tissue pO2 by EPRI following administration of METH in a mouse model. The successful execution of our proposed research will have tremendous impact on our ability to map tissue pO2 in the brain, and to understand the mechanistic significance of cerebral tissue pO2 variation in the pathophysiology of METH abuse providing new insights into the field of METH-induced neurotoxicity.